The present invention relates to a novel membrane-bound gluconate dehydrogenase (hereinafter referred to as xe2x80x9cGADHxe2x80x9d) from Erwinia cypripedii ATCC29267. More particularly, the present invention relates to a GADH, a DNA encoding the same, a recombinant plasmid containing said DNA, a host cell transformed with said recombinant plasmid, and the production of 2-keto-D-gluconate (hereinafter referred to as xe2x80x9c2KDGxe2x80x9d) from glucose or D-gluconate by culturing the recombinant cell.
Acetic acid bacteria, such as Erwinia, Glucobacter and Acetobacter, use alcohols and aldehydes as oxidizable substrates, converting it to acetic acid. Many carbohydrates, including glucose, glycerol, and sorbitol, and primary and secondary alcohols can also serve as energy sources, their oxidation characteristically resulting in the transient or permanent accumulation of partly oxidized organic products. This oxidation is mediated by membrane-bound dehydrogenases, such as alcohol dehydrogenase or aldehyde dehydrogenase, linked to the respiratory chain located in cytoplasmic membrane of the bacteria. There are two types of membrane-bound dehydrogenases: a quinoprotein and a flavoprotein having pyroroloquinoline quinon (PQQ) and flavin adenine dinucleotide (FAD), respectively, as cofactors. They are linked to the respiratory chain in the cytoplasmic membrane wherein electrons are transferred finally to oxygen, producing energy. Because the membrane-bound dehydrogenases can convert substrate outside the cells in addition to being of high activity for substrate, they have a significant advantage of being relatively high in substrate conversion rate and yield rate.
Much attention has been paid to the bioconversion processes using microorganisms on account that they have advantages over conventional chemical techniques, including economical and ecological aspects. In addition, the great advance which has been achieved in genetic recombination techniques and metabolic engineerings allows the bioconversion processes to overcome the conventional technical problems and to replace complicated chemical processes. The production of vitamin C is a representative example. In current, the production of 2-keto-L-gulonate, a precursor of vitamin C, via the sorbitol pathway or the glucose pathway, has been established or put to practical use.
In several species of the genera, Erwinia, Gluconobacter and Acetobacter, glucose is converted to gluconate, 2-keto-D-gluconate, and 12,5-diketo-D-gluconate by the mediation of glucose dehydrogenase, gluconate dehydrogenase and 2-keto-D-gluconate dehydrogase, respectively, which are linked to cytochrome C located in the cytoplasmic membrane of the bacteria (Ameyma et al., Agric. Biol. Chem. 51:2943-2950, 1987; Sonoyama et al., Agric. Biol. Chem. 52 : 667-674, 1988). 2,5-Diketo-D-gluconate is further converted to 2-keto-L-gulonate by 2,5-diketo-D-gulonate reductase (25DKG reductase). The above microorganisms, however, are disadvantageous tools in the aspect of the production yield of the vitamin C precursor because glucose undergoes both of the oxidative metabolism and the intracellular metabolism through which the intermediate products of the oxidative metabolism are transferrred inside the cell.
Accordingly, it is an object of the present invention to overcome the above problems and to provide a novel membrane-bound GADH form Erwinia cypripedii ATCC29267, which is useful for the production of 2KDG at high yields under the condition free of intracellular metabolism.
It is another object of the present invention to provide a novel DNA encoding the GADH.
It is a further object of the present invention to provide a novel recombinant plasmid containing the DNA and a host cell transformed with said recombinant plasmid.
In accordance with the present invention, 2KDG is converted at high yields from glucose or D-gluconate by culturing a recombinant cell, free of a ketogluconate metabolism, which harbors a plasmid containing a gene encoding the GADH.